Central to understanding how eukaryotic cells and the viruses that proliferate within them regulate the replication of their genomes is to understand the nature of DNA sites where replication begins and the proteins that interact with them. It now seems clear that while DNA alone contains many potential initiation sites for its replication, differentiated cells in higher eukaryotes select specific sites along their chromosomes to initiate DNA replication (DePamphilis (1996) DNA Replication in Eukaryotic Cells, Cold Spring Harbor Laboratory Press, NY.). The nature of these sites, the process by which they are established, and the mechanisms that regulate their activity remain critical questions. One advantage, however, to this complexity is the flexibility it offers the animal. Both the number of replication origins and their locations can be changed during development to meet the constantly changing requirements of gene expression and cell differentiation. In previous work, we developed various strategies for mapping the locations of initiation sites in the chromosomes of cells from metazoan animals to a resolution of less than or equal to 1 kilobase (kb), and used these methods to identify a specific origin of bidirectional DNA replication (OBR) about 17 kb downstream of the DHFR gene in hamster chromosomes. However, others detected replication bubbles throughout the 55 kb intergenic region downstream of this gene that suggested a random selection of initiation sites throughout a large initiation zone. During the past year, we have demonstrated directly that ori-b represents a primary replication origin in this genomic region by quantifying the relative number of initiation events throughout the initiation zone. We also discovered a second primary origin (ori-b') nearby that together with ori-b and ori-g, accounted for most of the replication bubbles detected by 2D gel electrophoresis in the DHFR gene initiation zone. Furthermore, we have been able to show that establishment of specific initiation sites, such as ori-b, depends upon the presence of specific DNA sequences, the formation of an intact nuclear structure, and changes that occur during G1-phase of the cell division cycle. However, initiation sites for DNA replication in mammalian chromosomes are not defined by their association with nucleoskeleton. Surprisingly, at least some active replication origins are associated with densely methylated clusters of CpG dinucleotides, and a reduction in DNA methylation results in loss of site-specific initiation. We also have cloned two of the mammalian homologues of yeast "origin recognition proteins" (ORC1, ORC2) and are in the midst of mapping the locations of ORC and "minichromosome maintenance" (MCM) proteins within the DHFR gene initiation zone. Both ORC and MCM proteins are believed to be required for establishing pre-replication complexes.